Microscopy utilizing the principle of Raman scattering has been expected to find application in the observation of cells in living organisms, etc., as a technique of molecular vibrational imaging that reflects molecular vibrational information.
Known microscopy techniques based on Raman scattering have heretofore included spontaneous Raman scattering microscopy and coherent anti-Stokes Raman scattering (CARS) microscopy.
In the former, the spectral information of the sample is acquired based on the fact that the frequency of the excitation light changes in accordance with the molecular vibrational frequency of the sample. However, since the resultant signal beam is extremely weak, improving the signal-to-noise (S/N) ratio has been a serious problem. On the one hand, in the latter, optical pulses of 2 colors (ωAS, ωS) are used to generate a CARS beam at a frequency (2ωAS−ωS) that is different from the excitation light. In CARS, the molecules are forced to oscillate, which has the advantage of obtaining a signal with a high S/N ratio. However, the problem is that the background presence of the so-called non-resonant signal, which is due to a non-linear response of electrons in the sample, degrades the contrast of the molecular vibrational images.
Stimulated Raman scattering (SRS) microscopy was proposed independently by the inventors (see Non-patent Document 1) and by Sunny Xie et al. of Harvard University (see Non-patent Document 2) as a way of addressing these contradictory problems of conventional Raman scattering microscopy.
The principle of stimulated Raman scattering microscopy is as follows.
Namely, the phenomenon of stimulated Raman scattering occurs at a focal spot when light is condensed on a sample in a state, in which one series of pulses of 2 colors (ωAS, ωS) is intensity-modulated, and the frequency difference between the 2 colors coincides with the molecular vibrational frequency of the sample at the focal spot. At such time, intensity modulation takes place in the excitation optical pulses that have not been intensity-modulated and the extent of intensity modulation due to the stimulated Raman scattering can be detected by photo-detecting the excitation light emitted from the sample. Therefore, molecular vibrational imaging of the sample is made possible based on the extent of the intensity modulation that takes place as a result of the stimulated Raman scattering.
FIG. 12 shows a block diagram of a conventional stimulated Raman scattering microscope used for principle confirmation.
As shown in FIG. 12, a conventional stimulated Raman scattering microscope 500 co-axially combines an anti-Stokes beam (ωAS), which is emitted from a titanium-sapphire laser 501, and a Stokes beam (ωS), which is emitted from an optical parametric oscillator 502 and intensity-modulated by an acousto-optic modulator (AOM) 503, with the help of a dichroic mirror 505 and condenses the beams through an objective lens 506 on a sample 507. At such time, scattered light is detected through an objective lens 508, a short-wave pass filter 509, and a focusing lens 510 using a photodiode (PD) 511 and a lock-in amplifier 512. It should be noted that the reference number 504 designates a mirror and the anti-Stokes beam (ωAS) has a repetition frequency of 76 MHz, a center wavelength of 765 nm, and a pulse width of 100 fs. In addition, the repetition frequency of the Stokes beam (ωS) is 76 MHz, its center wavelength 985-1005 nm, and its pulse width 200 fs. In addition, the frequency of the high-frequency signal source 513 is set to 2 MHz.